Methods and compositions for culturing a biological tooth

ABSTRACT

Tooth tissues include the pulp mesenchyme that forms the dentin and an epithelium that is responsible for enamel formation. Cells from these tissues were obtained from porcine third molars and were seeded onto a biodegradable scaffold composed of a polyglycolic acid-polylactic acid copolymer. Cell polymer constructs were then surgically implanted into the omentum of athymic nude rats so that the constructs would have a blood supply and these tissues were allowed to develop inside the rats. Histological analysis of 7.5 week-old implants revealed a dentin-like collagenous matrix containing hydroxyapatite mineral surrounding a core of mesenchymal cells that appeared analogous to pulp tissue. Infrequently, columnar epithelial cells were observed as a single layer on the outside of the dentin-like matrix similar to the actual arrangement of ameloblasts over dentin during early tooth development. Developing tooth tissues derived from such cell polymer constructs could eventually be surgically implanted into the gum of an edentulous recipient where the construct would receive a blood supply and develop to maturity, providing the recipient with a biological tooth replacement.

RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional ApplicationSer. No. 60/253,891, filed on Nov. 29, 2000.

BACKGROUND OF THE INVENTION

[0002] A developing molar tooth germ is encapsulated within the jaw fromwhich it will eventually erupt. The tooth germ is first observed as adeveloping bud (bud stage), which fans out into a cap-like structure(cap stage), and finally develops into a bell-like form (bell stage). Itis during the late bell stage that odontoblasts and ameloblastsdifferentiate and deposit the organic matrices of dentin and enamel. Ithas been well established that development of the tooth germ depends onreciprocal interactions between the epithelial and mesenchymal tissues(reviewed in: Thesleff et al., 1991)

[0003] Previously, Baba et al., (1996) have shown that molar tooth germsisolated from 16.5-day mouse embryos can be dissociated by enzymatictreatment. When the epithelial cells were separated from the mesenchymalcells, neither secreted enamel proteins nor cell proliferation wereobserved in either of the cultures. However, intriguingly, when thedissociated cells were cultured together, secretion of enamel proteinsand cell proliferation were observed. Furthermore, the dissociated cellsself-assembled back into a morphologically correct tooth germ that wassuccessfully cultured for more than 20 days. The authors hypothesizedthat since the tooth germ lacked a blood supply, its development wasprematurely terminated.

[0004] Tissue engineering is an interdisciplinary field that has evolvedfrom the combined expertise of life sciences and engineering principlesfor the creation of biological substitutes that maintain, restore, orimprove tissue function (Kim et al., 1999). Several tissues such asliver, intestine, bone, and cartilage have been successfully engineered(Kim et al., 1999). Dissociated cells from a tissue or organ have beenused to seed biodegradable polymer scaffolds, which are implanted withina suitable host such that a sufficient blood supply would allow thecells to organize into higher ordered structures around the scaffold.The maintenance of cell structures, such as those present in organs, isnot possible without a blood supply. Within a matter of weeks thescaffold dissolves and the dissociated cells will have organized into atissue or organ that was pre-determined by the size and shape of theoriginal scaffold. Tissue resembling small intestine, consisting of aneomucosa lined with smooth muscle, columnar epithelium, and gobletcells having villus-like structures, have been generated using the aboveapproach (Choi and Vacanti, 1997). Epithelial-mesenchymal cellinteractions are as essential for developing teeth as they are for theproper development of intestinal tissues. In the tooth, mesenchymalcells form the dentin while cells of epithelial origin form the enamel.Although each mineralized tissue is formed from its respective cells oforigin, epithelial-mesenchymal interactions are required to initiate themineralization process.

[0005] The demonstrated establishment of bioengineeredepithelial-mesenchymal cell-cell communications (intestine) and thesynthesis of mineralized tissues (bone and cartilage) necessary forgrowing teeth have already been accomplished. A significant need existsfor replacement teeth as observed from the common use of dental implants(year 2000 projected number of dental implant procedures was 910,000with a compound annual growth rate of 18.6% from 1998 to 2005, (AnnualIndustry Report, 2000). A biological tooth substitute that is properlyformed and integrated into the jaw of a human patient would outlastsynthetic dental implants since a living tooth responds to itsenvironment by migrating to maintain a proper bite, and has someregenerative properties in response to injury. Implants do not havethese capabilities. In addition, people who have genetically inheritedenamel (amelogenesis imperfecta) or dentin (dentinogenesis imperfecta)defects could be greatly helped by the availability of functional toothreplacements.

[0006]Amelogenesis imperfecta (AI) is a collection of genetic defectsmanifested by the malformation of dental enamel. One out of every 7,000to 14,000 children are affected (Backman and Holm, 1986; Chosack et al.,1979; Dummer et al., 1990; and Witkop Jr. and Sauk Jr., 1976). Bydefinition, the disorder must be limited to the dental apparatus andcannot be associated with more generalized defects (Witkop Jr. and Rao,1971).

[0007] Dentinogenesis imperfecta 1 (DGI1) is an autosomal dominantdental disease characterized by abnormal dentin production andmineralization (Xiao et al., 2001). Dentinogenesis imperfecta Sheildstype II (DGI-II) is also an autosomal dominant disorder in which boththe primary and permanent teeth are affected. It occurs with anincidence of 1:8,000 live births (Zang et al., 2001).

[0008] Recent advances in tissue engineering have demonstrated thatorgans derived from both epithelial and mesenchymal cells can befashioned into a pre-determined shape and size and can be provided witha blood supply (Choi et al., 1998; Choi and Vacanti, 1997).Specifically, small pieces (organoid units) of enzymatically digested6-day-old rat intestine were seeded onto sheets of non-wovenpolyglycolic acid (PGA) scaffolds and were incubated in culture forvarious times. Next, they were implanted into the omentum of syngeneicrats. The PGA provided the biodegradable three-dimensional scaffold andimplantation into the omentum provided the blood source. The organoidunits proliferated and generated larger complex cystic structures thatpossessed much of the morphology of the mature intestine. A key to thesuccess of the implants was not to delay the in vitro culture time morethan is necessary for the organoids to become firmly attached to thescaffold (Choi and Vacanti, 1997). Later, the engineered intestines werefurther characterized to show that they became phenotypically mature(Choi et al., 1998) and that successful anastomosis occurred between thetissue-engineered intestine and the native small bowel (Kaihara et al.,1999). Since as for the tooth, the intestine is also derived from theinteractions of both epithelial and mesenchymal tissues (Haffen et al.,1987), these data provide strong evidence that the dissociated toothgerm may also become fully mature through the techniques of tissueengineering.

[0009] A major difference between the tooth and the intestine is thatthe tooth becomes a mineralized tissue whereas the intestine does not.However, this is not a major technological difficulty since virtuallythe same tissue-engineering technique used to generated the intestinewas also used to engineer mineralized phalanges with joints (Isogai etal., 1999). The phalanges were specifically designed to have a humanshape and were shown to possess mature articular cartilage andsubchondral bone. Thus, we are generating a tissue-engineered tooth byusing techniques similar to those that were used successfully togenerate an intestine and phalanges with joints.

[0010] The practice of dentistry would be revolutionized, by providingthe patient and oral surgeon a means to replace a defective or diseaseddentition with a healthy and permanent biological dentition. Thesestudies could yield new insight into the regulation of enamel formationand may provide a means of generating tissue engineered dentin or enamelmaterials that could be used to repair unhealthy teeth.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 shows tooth scaffolds.

[0012]FIG. 2 shows tooth scaffolds-PGLA.

[0013]FIG. 3 shows scanning electron micrographs of a PLGA scaffold plussalt.

[0014]FIG. 4 shows scanning electron micrographs of a PLGA scaffold plussugar.

[0015]FIGS. 5 and 6 show removal of a porcine third molar.

[0016]FIG. 7 shows porcine tooth tissue culture.

[0017]FIG. 8 shows tissue culture with a Von Kossa stain.

[0018]FIG. 9 shows rat radiographs of a human tooth.

[0019]FIG. 10 shows rat radiographs of an implant, 7½ weeks.

[0020]FIGS. 11 and 12 show dissection of tissue.

[0021]FIGS. 13 and 14 show dissected tooth tissue cysts, 7½0 weeks.

[0022]FIG. 15 is a schematic drawing of tissue sectioning.

[0023]FIG. 16 shows sectioned tissue with Goldner's stain.

[0024]FIG. 17 shows a cell seeded incisor scaffold 20 weekspost-implantation.

[0025]FIG. 18A shows a histological section of a 20-week tooth budstained with hematoxylin and eosin and then counterstained by the methodof Von Kossa.

[0026]FIG. 18B shows the root tip of the bud of FIG. 18A, showingcolumnar odontoblasts and Hertwig's root sheath.

[0027]FIG. 19A shows an engineered tooth with dentin, enamel, andameloblasts stained with hematoxylin and eosin.

[0028]FIG. 19B shows an engineered tooth with dentin, enamel, andameloblasts stained by Goldner's method.

[0029]FIG. 20A shows a histological section of a 30-week implant stainedwith hematoxylin and eosin, having demineralized enamel interior to thedentin.

[0030]FIG. 20B shows an ameloblast cell layer adjacent to enamel spaceof the implant of FIG. 20A.

[0031]FIG. 20C shows the cementum of the implant of FIG. 20A withembedded nuclei of putative cementoblasts.

DESCRIPTION OF THE INVENTION

[0032] Our goal is to produce a biological tooth replacement usingtissue-engineering methodology based on seeding dissociated tissues ontobiodegradable polymer scaffolds, and allowing the cell/polymerconstructs to develop into tooth tissues inside of a suitable host.Polymer scaffolds are molded in the shape of human teeth usingpolyvinylsiloxane molds and seeded with dissociated tissues fromunerupted porcine third molars. Cell/polymer constructs are implantedinto the omentum of athymic rats so that the developing tooth tissuesreceive an adequate blood supply. Cells dissociated from the enamelorgan and pulp organ and cells from tissue cultures derived from toothtissues, are seeded onto molded tooth-shaped polymers, and implanted fordevelopment in rat hosts. Analysis of the resulting tooth tissues isperformed using histological staining methods such as Von Kossa(calcification), Goldner's (ossification), and Van Gieson's (collagen).Immunohistochemical staining is also performed using antibodies specificfor tooth epithelial markers (keratin, amelogenin) and mesenchymalmarkers (osteocalcin, bone sialoprotein and dentin sialophosphoprotein).The results of these experiments establish the identity of ameloblaststhat are responsible for enamel formation, and odontoblasts that areresponsible for dentin formation, within the engineered tooth tissues.Immunofluorescence using the above markers is applied to cells inculture to characterize them prior to seeding on polymer scaffolds. Insitu hybridization is used to detect the presence of DSPP mRNA, a markerfor odontoblast cells, and to help distinguish between tissues of therat host and developing porcine tooth tissues.

[0033] Table 1 provides an overview of the invention. Table 2 providesan overview of the polymer scaffold preparation. TABLE 1 ExperimentalApproach Remove porcine third molar Surgical implantation of seededpolymers Mince tissue and treat Incubation in rat omentum enzymaticallyMild mechanical dissociation X-ray rats Count cells Dissect tooth tissuePolymer seeded with cells and Histology “organoids”

[0034] TABLE 2 Polymer Scaffold Preparation Polyglycolic acid (PGA),poly-L-lactic acid (PLLA), polylactic-co- glycolic acid (PLGA) Dissolvepolymer in chloroform or dioxane NaCl or glucose crystals added toincrease porosity (PLGA scaffolds only) Combine polymer solution andcrystals in tooth mold Freeze-dry to evaporate solvent Place scaffold inwater to dissolve salt/sugar

EXAMPLE

[0035] A. Materials and Methods

[0036] Chemicals. polyglycolic acid (PGA), poly-L-lactic acid (PLLA),poly-L-lactide-co-glycolide (PLGA), chloroform, dichloromethane,polyvinylsiloxane dental impression material (Reprosil), sodiumchloride, Hank's Balanced Salt Solution (HBSS), phosphate-bufferedsaline solution (PBS), Dulbecco's Modified Eagle Medium (DMEM), fetalbovine serum, Glutamax, penicillin, streptomycin, sorbitol, 0.9% salinesolution, iodine solution (Povidine), 70% ethanol, collagen (type I),0.01 M hydrochloric acid, collagenase, dispase, ketamine, xylazine(Rompun).

[0037] Tissues. Human incisors and molars, six-month-old porcine thirdmolar tooth tissue.

[0038] Preparation of tooth molds. Extracted human incisors and molarswere used to cast negative impression tooth molds in polyvinylsiloxanedental impression material (Reprosil). Once the impression materialhardened, the teeth were removed by cutting an opening in one side ofthe mold with a razor blade. This method leaves a tooth-shaped cavityinside the impression material which can be filled with polymer solutionfor the preparation of biodegradable tooth scaffolds.

[0039] Preparation of polymer tooth scaffolds. PGA mesh material wasbroken up into 1-2 mm flakes and packed into the cavity of a tooth moldto fill it completely. The remainder of the cavity volume was filledwith a 3% w/w PLLA solution in chloroform. The PGA/PLLA mixture washeated to approximately 400° F. for 5 minutes to bond the two polymersand then lyophilized for 48 h. For PLGA tooth scaffolds, PLGA crystalswere first dissolved in chloroform to 5% w/w. Negative tooth molds werepacked to half-capacity with sodium chloride crystals (75-150 μm) andthe remainder of the mold volume was filled with 5% PLGA solution.Sodium chloride crystals were added to create a thick slush in the PLGAsolution and the mixture was lyophilized for 48 h. Scaffolds wereremoved from the molds and placed in distilled water for 24 h to leachout the salt crystals leaving behind a porous PLGA sponge material inthe shape of a tooth.

[0040] Tissue dissociation. Fresh pig jaw dissected from a freshlyslaughtered six-month-old pig was placed on ice for transport. The jawwas split in two and muscle and connective tissue were removed from thebone using a razor blade. A dental drill fitted with a spherical bit wasused to drill holes along the lingual side of the bone surface alonglines adjacent to the regions harboring the 3^(rd) and 2^(nd) uneruptedmolars. Bone chisels were used to break the bone in between the drilledholes and then the resulting bone flap was pried and lifted away toexpose the unerupted molars. A dental probe was used to carefully liftout the 3rd molar and connective tissue was cut with surgical scissors.The molars were placed in ˜50 ml of Hank's balanced salt solution (HBSS)and kept at 4° C. in 50 ml sterile conical tubes.

[0041] Prior to mincing the tissues, the immature tooth cusps wereremoved and discarded. The remaining enamel and pulp organ tissues wereminced into 2-3 mm³ pieces in a sterile Petri plate in HBSS. Tissueswere washed 5 times in HBSS, minced into <1 mm³ pieces and then treatedwith 1.5 units of Vibrio alginolyticus collagenase and 12 units ofBacillus polymyxa dispase for 25 minutes at room temperature. Gentlemechanical dissociation of tissues was achieved by pipetting thesuspension up and down in a 25 ml pipette for 10 min followed by 15 minwith a 10 ml pipette. Tissues were washed five times in DMEM (containing2.5% FBS, 2% sorbitol, Glutamax, 50 units/ml penicillin, 50 □g/mlstreptomycin) and then cells were counted using a hemacytometer. Typicalcell yields were 2.0×10⁶ cells/ml.

[0042] Seeding of biodegradable polymer scaffolds. PGA/PLLA and PLGAtooth scaffolds were coated with collagen overnight at 4° C. in a 1mg/ml type I collagen solution in 0.01 M HCl. Scaffolds were washedthree times in PBS then three times in DMEM+supplements (see above).˜2.0×10⁶ cells were seeded onto each tooth scaffold and cells were givenat least 1 hour to attach. Laparotomies were performed on athymic nuderats and seeded scaffolds were implanted into the omentum to provide ablood source for the developing tooth tissues. Tissues were allowed todevelop inside the host animals for 7-20 weeks before they weresacrificed and the engineered tissues harvested.

[0043] B. Results

[0044] The results of these experiments are generally summarized inTable 4. TABLE 4 Tooth Tissue Engineering Schedule Time Date MaterialShape (weeks) Status Aug. 10, 2000 Poly-glycolide Short tube 8.5Sacrificed (PGA) Long tube Sacrificed Human Alive incisor Aug. 23, 2000PGA + Incisor 6.5 Deceased poly-L-lactide Incisor Alive (PLLA) MolarAlive Sep. 7, 2000 Poly-L-lactide-co- Molar 4.5 Alive glycolide IncisorAlive (PLGA) + salt crystals Sep. 27, 2000 PLGA + Molar — Deceased saltor sugar crystals Molar Deceased Incisor Deceased Incisor Deceased

[0045] Polymer Tooth Scaffolds. Human incisors and molars were used tomake negative impressions in Reprosil dental impression material. PGAmesh material was then broken into flakes and these flakes were used tocompletely fill the tooth mold. The remainder of the mold cavity volumewas filled with a 3% PLLA solution in chloroform. The PGA/PLLA mixturewas heated to approximately 400° F. for 5 minutes to bond the twopolymers and then lyophilized for 48 hours. The PLGA tooth scaffoldswere made as described in the Materials and Methods section.

[0046] Tooth Tissues. Immediately after slaughter, mandibles fromsix-month old pigs were collected at the slaughterhouse and transportedon ice to the laboratory. The second and third unerupted molars fromeach hemimandible were dissected from the jawbone and were immersed inseparate vials containing Hanks balanced salt solution. All teeth werekept at 4° C. prior to dissociation of the tissues. The enamel and pulporgan tissues were minced, washed, minced again, and treated withcollagenase/dispace in order to obtain the greatest amount of singlecells in suspension. The cells were then washed several more times andwere resuspended in DMEM containing 2.5% FBS and 2% sorbitol.

[0047] Seeding of Biodegradable Polymer Scaffolds. PGA/PLLA and PLGAtooth scaffolds were coated with collagen overnight at 4° C. in a type Icollagen solution in 0.01 M HCl. Scaffolds were washed three times inPBS then three times in DMEM plus FBS and sorbitol. Cells were seededonto each tooth scaffold and were given at least 1 h to attach.Laparotomies were performed on athymic nude rats and seeded scaffoldswere implanted into the omentum to provide a blood source for thedeveloping tooth tissues. The tissues were allowed to develop inside thehost animals for 7-20 weeks before they were sacrificed and theengineered tissues harvested.

[0048] Characterization of Bioengineered Tooth Tissues. Host animalswere sacrificed after 7.5 weeks of development and the tooth tissueswere dissected, preserved and fixed in formalin, and embedded inparaffin for histological sectioning. Tissue sections were stained withhematoxylin and eosin and counterstained by the Von Kossa method toidentify mineralized tissues. Tissues were also stained by the method ofvan Gieson to identify areas of ossification and were stained by themethod of Goldner to detect the presence of collagen.

[0049] Histological sections of engineered tooth tissues revealed anorganization analogous to the early tooth bud. Present was a layer ofcollagenous matrix that appeared similar to that observed in dentin orbone. The surrounding region of mesenchyme looked like what is observedin pulp tissue. Infrequently, a single layer of columnar epithelium wasobserved on the outer face of the collagenous matrix, resemblingepithelial ameloblast cells, which form dental enamel. Some regions ofthe collagenous matrix stained positively for the presence of calcifiedmineral deposits suggesting that biomineralization had occurred.

[0050] C. Discussion

[0051] Preliminary results demonstrate successful use of porcineodontogenic cells to generate replacement molar and incisor teeth.Mineralization was observed in a four week-old tissue culture of thedissociated porcine third molar tissues, suggesting that a mixture ofdissociated tooth tissue cells can spatially reorganize themselves invitro, and generate calcified deposits. Dissociated porcine tooth tissuecells were seeded onto collagen-coated PGA scaffolds and implanted intothe omenta of rat hosts. Histological analysis of 7.5-week-old implantedPGA scaffolds revealed an organization similar to that of the earlytooth bud. A layer of collagenous matrix similar to dentin or bonesurrounded the mesenchyme tissue. This resembles what naturally occursin pulp tissue. Rarely, a single layer of columnar epithelium wasobserved on the outer face of the collagenous matrix. This is similar tothe organization of enamel-forming epithelium just prior to theformation of dental enamel. The presence of calcified mineral depositssuggests that biomineralization had occurred. These results demonstratethat it is possible to grow mineralized tooth tissues usingbiodegradable polymer tooth scaffolds seeded with tooth bud cells. Thus,we have demonstrated that by use of the tissue engineering techniquesdescribed here, it is possible to grow mineralizing tissues thatresemble those of the developing tooth.

[0052] Preparation of Tooth Molds.

[0053] Rationale. Tooth molds are used to prepare scaffolds in theshapes of individual human teeth so that the seeded tooth cells willform a tooth of a predetermined shape and size.

[0054] Experimental approach. Extracted human incisors and molars areused to create tooth molds in polyvinylsiloxane dental impressionmaterial (Reprosil). Once the impression material has hardened, theteeth are removed by cutting an opening in one side of the mold with arazor blade. This method leaves a tooth-shaped cavity inside theimpression material, which can be filled with polymer solution for thepreparation of biodegradable tooth scaffolds.

[0055] Preparation of Polymer Tooth Scaffolds.

[0056] Rationale. Polymer scaffolds of optimal porosity are necessary sothat the seeded cells can migrate through the scaffold to theirappropriate positions to begin forming the tissue-engineered tooth.

[0057] Experimental approach. PGA mesh material is broken into 1 mm²flakes and packed into the cavity of a tooth mold to fill it completely.The remainder of the cavity volume is filled with a 3% w/w PLLA solutionin chloroform. The PGA/PLLA mixture is heated to approximately 400° F.for 5 min to bond the two polymers and then is lyophilized for 48 h. ForPLGA tooth scaffolds, PLGA crystals will first be dissolved inchloroform to 5% w/w concentration. Tooth molds are packed tohalf-capacity with sodium chloride crystals (75-150 μm) and theremainder of the mold volume is filled with 5% PLGA solution. Sodiumchloride crystals are added to create a thick PLGA slush, and themixture is lyophilized for 48 h. Scaffolds are removed from the moldsand placed in distilled water for 24 h to dissolve salt crystals,leaving behind a porous PLGA sponge material in the shape of a tooth.

[0058] Isolation and Preparation of Porcine Tooth Tissues for Seedingonto Biodegradable Polymer Scaffolds.

[0059] Rationale. Optimized procedures for dissociating tooth tissuesfor seeding onto the polymer scaffold are necessary so that both theepithelial and mesenchymal cells will attach, migrate to theirappropriate positions, and form their respective mineralized tissues(enamel and dentin).

[0060] Experimental approach. A jaw dissected from a freshly slaughteredsix-month old pig is placed on ice for transport from the farm (Athol,Mass.) to The Forsyth Institute. The jaw is split at the midline. Muscleand connective tissue are removed from the bone using a razor blade. Adental drill fitted with a spherical burr is used to drill holes alongthe jaw (lingual side) surrounding the region of the M2 and M3 uneruptedmolars. Bone chisels are then used to break the bone in between thedrilled holes, and the resulting bone flap is removed to expose theunerupted molars. A dental probe is used to carefully lift out M2 and M3tooth sacs, and the connective tissue is removed with surgical scissors.The molars are placed in 50 ml of Hank's balanced salt solution (HBSS)and kept at 4° C. in 50 ml sterile conical tubes. When present, immaturetooth cusps are removed and discarded, and the remaining enamel and pulporgan tissues are minced into 2-3 mm³ pieces in a sterile Petri dish inHBSS. Tissues are washed 5 times in HBSS, minced into <1 mm³ pieces andtreated with 1.5 units of Vibrio alginolyticus collagenase and 12 unitsof Bacillus polymyxa dispase for 25 min at room temperature. Gentlemechanical dissociation of tissues is achieved by gentle pipetting witha 25 ml pipette for 10 min followed by 15 min with a 10 ml pipette. Thedispersed cells are washed five times in DMEM (containing 2.5% FBS, 2%sorbitol, Glutamax, 50 units/ml penicillin, 50 μg/ml streptomycin) andpelleted by gentle centrifugation at approximately 400×g and countedusing a hemacytometer. Typical cell yields are approximately 5.0×10⁶cells/ml.

[0061] Molded PGA/PLLA and PLGA tooth scaffolds are coated with collagenovernight at 4° C. in a 1 mg/ml type I collagen solution in 0.01 M HCl.Scaffolds are washed three times in PBS then three times inDMEM+supplements (see above). Approximately 2.0×10⁶ cells are seededonto each tooth scaffold and allowed to attach for at least 1 hour.Laparotomies are performed on athymic nude rats and the seeded scaffoldsare implanted into the omentum, providing a blood source for developingtooth tissues. The implants are allowed to develop inside the hostanimals for 7-35 weeks before the engineered tissues are harvested.

[0062] To our knowledge, no study has ever examined the feasibility ofgrowing biological teeth using dissociated tooth tissues seeded onbiodegradable polymer scaffolds. One group has usedhydroxyapatite/tricalcium phosphate powder mixed with cultured dentalpulp cells to generate a small amount dentin matrix secreted byodontoblast-like cells six weeks after subcutaneous implantation in nudemice (Gronthos et al., 2000). However, using our approach we haveobtained structures resembling developing and mature teeth with dentinsecreted by odontoblasts, enamel secreted by ameloblasts, a well-definedpulp chamber and putative cementoblasts embedded in a cementum matrix(see Preliminary Data). Thus, our approach has demonstrated that it ispossible to engineer developmentally advanced tooth tissues.

Preliminary Data

[0063] In vitro analysis of dissociated tooth tissues. Six-month oldporcine third molars (M3) were dissociated into cell suspensions andgrown in culture for a period of four weeks. The cell cultures exhibitedextensive mineralization, as measured by Von Kossa staining (data notshown), suggesting that the dissociated tooth tissues could spatiallyreorganize themselves in vitro to form calcified deposits.

[0064] Analysis of Seeded and Implanted Biodegradable Scaffolds.

[0065] Twenty week implant. Dissociated enamel and pulp cells obtainedfrom a 6 month-old pig third molar were seeded onto a PGA scaffoldmolded in the shape of a human incisor of approximately 1 cm by 0.5 cmin size. The cell/polymer construct was implanted into the omentum of anude rat host and allowed to develop for 20 weeks. At this time,histological analysis revealed small tooth-shaped tissues within theimplant, which were similar in appearance to that of a very small cusptip (FIG. 17). We observed mineralized dentin-like tissue (D) andbeneath the dentin, was a pre-dentin-like layer (PD) that appeared to besecreted by odontoblast-like cells (0). Vascularized mesenchymeresembling that of pulp tissue (PT) filled the remainder of the pulpcavity (FIG. 17).

[0066] The cellular organization of another 20 week implant clearlyresembles that of an early bell stage tooth bud (FIGS. 18A-B). The toothtissue was ˜2 mm in diameter and exhibited distinct coronal and apicalorganization, with recognizable cusps and root tips. Putativeodontoblasts (0) lined the inner surface of an apparently collagenousdentin matrix (D) (FIGS. 18A-B) and putative Hertwig's root sheathepithelia (H) was also present adjacent to the developing root tips(FIG. 18B).

[0067] In summary, the 20-week tooth tissues contained putativepre-dentin and mineralized dentin components, and vascularizedmesenchymal cells resembling pulp tissue populated the pulp chamber. Inthis twenty-week implant no ameloblast-like epithelial cells wereobserved on the outer face of the putative dentin tissue.

[0068] Twenty-five week implant. A tooth tissue implant consisting of aPGA polymer scaffold molded in the shape of a human incisor seeded with˜2.0×10⁶ porcine tooth tissue cells was dissected from a nude rat at 25weeks post implantation. The tissue was fixed in neutral formalin,embedded in paraffin, sectioned and then stained with hematoxylin andeosin (FIG. 19A). A tooth bud with a diameter of 2 mm was discoveredwithin the excised tissue. The interior core of the tooth bud consistedof pulp-like mesenchymal cells lined with columnar odontoblasts whichwere adjacent to a dentin-like layer, as has been observed in previous20-week tooth buds (see FIGS. 17 and 18A-B). In some locations adifferent mineralizing layer (E, EM) was observed (FIG. 19A) whichclosely resembled decalcified porcine enamel. A darkly stained regionwas found directly adjacent to numerous columnar cells possessingpolarized nuclei which closely resembled ameloblasts (A). This denselystained region was thought to be enamel matrix since the staining wassignificantly reduced in the deeper layers just as it is for naturallyforming enamel. The same tissue was stained by the method of Goldner(FIG. 19B) which stains osseous tissues blue-green while the maturedental enamel stains a bright red color (Dr. Ziedonis Skobe, personalcommunication) Thus, after 25 weeks of implant development, we haveobtained engineered tissues that are composed of the two majormineralizing structures of the tooth: the dentin and enamel.

[0069] Thirty week implant. Anatomy of an inverted tooth. Porcine toothtissues were seeded onto a PLGA scaffold which was implanted into a nuderat and harvested 30-weeks later. FIGS. 20A-C show a demineralized,hemotoxylin-stained section from the implant. FIG. 20A shows a layer ofdentin that surrounds a thick layer of enamel. A close-up reveals theunmistakable columnar rows of ameloblasts (A) with polarized nuclei(FIG. 20B). The cellular tissue adjacent to the ameloblasts ismorphologically similar to the stratum intermedium and the remainingcellular tissue is very similar to the stellate reticulum. Thus, itappears that the three major tissue morphologies of the enamel organ arealso present within this inverted tissue engineered tooth. Even morestriking, was the appearance of putative cementoblasts (C) that wereembedded within their own matrix (FIG. 20C).

[0070] Thus, although this is an inverted tissue-engineered tooth, thistooth appears to be developing all the necessary components of a healthymaturing tooth. We believe that this small tooth structure(approximately 2 mm in length) is inverted because not enough cells wereoriginally seeded onto the scaffold. Previous studies demonstrate thatapproximately, 20-50 million cells are required per square cm forengineered tissues to conform to the shape of the scaffold and thisimplant had approximately 10 fold less cells than was required.

[0071] In a separate set of experiments designed to confirm the identityof the engineered tissues, we performed immunohistochemical analysisusing antibodies specific for proteins present in epithelia,dentin/bone, or enamel. Immunohistochemical analysis of decalcifiedporcine M3 control teeth with an anti-pancytokeratin antibody resultedin staining of porcine ameloblasts and stratum intermedium cells, but nostaining of the odontoblasts. The same antibody reacted relativelystrongly with rat epithelial cells present in sectioned rat mandibletissue. We are evaluating engineered tooth tissues byimmunohistochemical staining with the anti-pancytokeratin antibody aswell as antibodies against amelogenin, osteocalcin, bone sialoprotein,and dentin sialophosphoprotein (DSPP). We are currently performing insitu hybridization analysis for the dentin-specific protein DSPP toconfirm the identity of the odontoblasts and dentin tissues. Thesemarker analyses will help identify ameloblasts, odontoblasts, andcementoblasts present in the tissue-engineered tooth tissues.

[0072] In conclusion, we have demonstrated successful engineering ofrecognizable teeth, using biodegradable polymer scaffolds seeded withporcine third molar tooth cells. The teeth form dentin from cellsappearing to be odontoblasts, have a well defined pulp chamber, possessputative Hertwig's root sheath epithelial, possess putativecementoblasts, and have a morphologically correct enamel organconsisting of stellate reticulum, stratum intermedium, and ameloblasts,and have what appears to be fully formed dental enamel.

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1. A method of generating tooth tissue comprising: applying tooth germcells onto a biodegradable polymer scaffold and allowing the tooth germcells to develop into a tooth.
 2. The method of claim 1, furthercomprising: forming a tooth mold containing the biodegradable polymerscaffold.
 3. The method of claim 1, wherein the biodegradable polymerscaffold has a shape of a tooth.
 4. The method of claim 3, wherein thebiodegradable polymer scaffold has a shape of a human tooth.
 5. Themethod of claim 1, wherein the tooth germ cells are mammalian.
 6. Themethod of claim 1, wherein the tooth germ cells are porcine.
 7. Themethod of claim 1, wherein the tooth germ cells comprise one or more ofcells selected from the group consisting of cells from an enamel organ,cells from a pulp organ and tissue cultured cells derived from a toothtissue.
 8. The method of claim 1, wherein the tooth germ cells areapplied to the biodegradable polymer scaffold with between about 20 to50 million cells per square centimeter of scaffold.
 9. The method ofclaim 1, wherein the biodegradable polymer scaffold is selected from thegroup consisting of poly(lactide), poly(glycolide) andpoly(L-lactide-co-glycolide).
 10. The method of claim 1, wherein thebiodegradable polymer scaffold is coated with collagen prior to saidapplying.
 11. The method of claim 1, further comprising: implanting thescaffold into an omentum of a host mammal.
 12. The method of claim 11,wherein the host mammal is a rat.
 13. A method of generating toothtissue comprising: forming a biodegradable polymer scaffold; applyingtooth germ cells onto the biodegradable polymer scaffold; and implantingthe scaffold to which tooth germ cells have been applied into a hostanimal.
 14. The method of claim 13, further comprising: preparing atooth mold, wherein the biodegradable polymer scaffold is formed in thetooth mold.
 15. The method of claim 13, wherein the biodegradablepolymer scaffold is in a shape of a tooth.
 16. The method of claim 15,wherein the biodegradable polymer scaffold is in a shape of a humantooth.
 17. The method of claim 13, wherein the tooth germ cells aremammalian.
 18. The method of claim 13, wherein the tooth germ cellscomprise one or more of cells selected from the group consisting ofcells from an enamel organ, cells from a pulp organ and tissue culturedcells derived from a tooth tissue.
 19. The method of claim 13, whereinthe tooth germ cells are applied to the biodegradable polymer scaffoldwith between about 20 to 50 million cells per square centimeter ofscaffold.
 20. The method of claim 13, wherein the biodegradable polymerscaffold is selected from the group consisting of poly(lactide),poly(glycolide) and poly(L-lactide-co-glycolide).
 21. The method ofclaim 13, wherein after applying, the tooth germ cells are allowed toattach to the scaffold for at least one hour prior to said implanting.22. The method of claim 13, wherein the biodegradable polymer scaffoldis coated with collagen prior to said applying.
 23. The method of claim13, wherein said implanting is into an omentum of the host mammal.
 24. Atooth produced from tooth germ cells and a biodegradable polymerscaffold.
 25. The tooth of claim 24, wherein the tooth is in a shape ofa human tooth.
 26. The tooth of claim 24, wherein the tooth germ cellscomprise one or more of cells selected from the group consisting ofcells from an enamel organ, cells from a pulp organ and tissue culturedcells derived from a tooth tissue.
 27. The tooth of claim 24, whereinthe biodegradable polymer scaffold is selected from the group consistingof poly(lactide), poly(glycolide) and poly(L-lactide-co-glycolide). 28.The method of claim 1, wherein said tooth germ cells are human.
 29. Themethod of claim 1, further comprising: implanting said tooth into thegum of a host mammal.
 30. The method of claim 13, wherein saidimplanting is into a mouth of the host mammal.